Cotton is a dual purpose crop, producing both fiber and seed as valuable primary agricultural products. Normally, cottonseed products, including hulls (26%), linters (9%), oil (16%) and cottonseed meal (45%) represent approximately 15% of the farm value of the cotton crop (Cherry and Leffler, Seed. In “Cotton, agronomy monograph No. 24” (eds R J Kohel, C F Lewis) pp. 511-569. Crop Science Society of America, Madison, Wis., USA, 1984), while lint provides most of the remaining 85% of the value. Cottonseed oil is the most valuable product derived from cottonseed.
Cottonseed oil has a long tradition of use in food processing. Since cottonseed oil has a bland, neutral flavor that does not mask the inherent flavor of food, it is a popular and widely used oil for deep frying in the snack food and food service sector (Jones and King, Cottonseed Oil. National Cottonseed Products Associations, Inc. and the Cotton Foundation, Memphis, Tenn., USA, 1993). Cottonseed oil is also commonly used as an ingredient in marinades, dressings, pastries, margarines, and shortenings.
The nutritional and industrial value of cottonseed oil, like other vegetable oils, is affected by the composition of fatty acids in the oil, ie. the relative level of each the fatty acids in the oil, and the properties conferred by the carbon chain length and level of unsaturation of each fatty acid.
Isolated and purified cottonseed oil is composed mostly (>95%) of triacylglycerols (TAGs) that are synthesized and deposited during seed development. TAG molecules consist of three fatty acids esterified to a glycerol backbone, designated the sn-1, sn-2 and sn-3 positions. Briefly, the de novo biosynthesis of fatty acids in cotton seed, as in other oilseeds, occurs in the stroma of plastids during development and growth of the seeds, ie. before maturation. Fatty acids are then exported from the plastids in the form of acyl-CoA thioesters to the cytoplasmic endomembrane systems (endoplasmic reticulum, ER) where modification of fatty acids occurs after transfer of the acyl groups from the CoA thioesters to phospholipids by acyltransferases. This is followed by TAG assembly and storage in the oleosomes.
The biotin-containing enzyme acetyl-CoA carboxylase (ACCase) catalyses the first committed step in the pathway by activating acetyl-CoA to the three carbon intermediate, malonyl-CoA, by addition of a carboxyl group. The malonyl group is then transferred from CoA to an acyl-carrier protein (ACP), which serves as the carrier for the growing fatty acid chain. Malonyl-ACP is reacted with a second acetyl-CoA condensing enzyme, ketoacyl-ACP synthase III (KASIII), resulting in a four carbon chain. The repeated process of adding two-carbon units on the elongated fatty acid chain is catalyzed by KASI leading to the formation of palmitoyl-ACP. KASII catalyzes the elongation of palmitoyl-ACP to stearoyl-ACP. A soluble stearoyl-ACP Δ9-desaturase introduces the first double bond into stearoyl-ACP to convert it to oleoyl-ACP in the plastid. The extended, saturated fatty acyl chain and the monounsaturated oleate are cleaved off the ACP by a specific thioesterase enzyme, FatB or FatA, respectively, enabling them to exit the plastid and enter the cytoplasm. Saturated fatty acids released into the cytoplasm are not further modified. However, oleic acid can be further modified on the endoplasmic reticulum (ER) membranes by the action of membrane-bound desaturases. Phosphatidylcholine (PC)-bound acyl chains serve as a substrate for ER localized, lipid modifying enzymes, such as fatty acid desaturase 2 (FAD2) which introduces a double bond into oleic acid on the sn-2 position of PC to produce linoleic acid. All the modified and unmodified fatty acyl groups then form a pool while attached to CoA. In cotton, but not in other temperate zone oilseeds, oleic acid may be used as substrate for cyclopropanation catalysed by cyclopropane fatty acid synthase to produce dihydrosterculic acid. This fatty acid is subsequently desaturated to produce sterculic acid and then α-oxidased to produce malvalic acids. Finally fatty acyl groups are incorporated into storage lipids via the Kennedy pathway by the sequential esterification of glycerol-3-phosphate by the action of a series of TAG assembly enzymes.
The enzyme acyl-ACP thioesterase (FatB) (EC 3.1.2.14) catalyses the hydrolysis of the thioester bond between the acyl moiety and ACP in acyl-ACP and the release of free fatty acid in the plastid. The free fatty acid is then re-esterified to CoA in the plastid envelope as it is transported out of the plastid. FatB belongs to a class of nuclear encoded, soluble, fatty acid thioesterase (FAT) enzymes which are first translated as a precursor proteins. The substrate specificity of the FAT enzymes in the plastid is therefore involved in determining the spectrum of chain length and degree of saturation of the fatty acids exported from the plastid. FAT enzymes can be classified into two classes based on their substrate specificity and nucleotide sequences, FatA and FatB (Jones et al., Plant Cell 7: 359-371, 1995). FatA prefers oleoyl-ACP as substrate, while FatB shows higher activity towards saturated acyl-ACPs of different chain lengths. Genes encoding FatB enzyme were first isolated from plant species accumulating medium chain-length saturated fatty acids such as lauric acid (C12:0) from California bay tree (Umbellularia californica). Overexpression of the U. californica FatB1 gene in transgenic canola led to production of high-laurate oil with laurate comprising 50% of the total fatty acids in the oil (Voelker et al., Plant Journal 9: 229-241, 1996). Subsequent studies demonstrated that several FatB orthologues are present in tissues of plants, including in seeds, with substrate specificity ranging from C8:0-ACP to C18:0-ACP.
Cottonseed oil produced by conventional (wild-type) upland cotton (G. hirsutum) typically contains approximately 26% palmitic acid (range 22-28%), 2% stearic acid, 15% oleic acid (range 13-18%) and 58% linoleic acid (range 52-60%) (Cherry, J. Am. Oil Chem. Soc. 60: 360-367, 1983; O'Brien, Cottonseed Oil. In: F. D. Gunstone (Ed.) Vegetable Oils in Food Technology: Composition, Properties and Uses. Blackwell Publishing, Oxford, pp. 203-230, 2002). In addition to these fatty acids which are also present in most other temperate zone oilseed crops, unhydrogenated cottonseed oil also contains low levels (0.5-1%) of cyclopropane or cyclopropene fatty acids, mainly malvalic, sterculic and dihydrosterculic acids (Shenstone and Vickery, Nature 190: 68-169, 1961; Cherry, 1983 (supra)). Cyclopropane (CPA) and cyclopropene (CPE) fatty acids are found in the seed oils of plants in the order of Malvales and some gymnosperms, including in cotton. Two Sapindaceae species, Lychee (Litchi chinensis) and Longan (Euphoria longan) contain up to 40% CPA fatty acids in their seedoils, mostly as dihydrosterculic acid (DHS). The seed oil of Sterculia foetida contains up to 78% CPE fatty acids, mainly sterculic (STC) and malvalic acids (MVL). Unhydrogenated cottonseed oil contains relatively small amounts (0.5-1.0%) of CPE and CPA fatty acids, mostly in embryo axes. Cotton roots and hypocotyls are also found to accumulate low levels of STC and MVL. CPA and CPE are not found at detectable levels in major oilseed crops other than cotton, including in palm oil, soybean, corn, canola, mustard, sunflower, safflower, peanut, linseed, other Brassicas etc.
The First committed step to produce these uncommon fatty acids is catalysed by a cyclopropane fatty acid synthase (CPA-FAS) which adds a methylene group across the double bond of oleic acid to produce DHS (FIG. 2). In cotton, most of the DHS is desaturated by the enzyme CPA desaturase to produce STC, most of which is further modified by α-oxidation to form MVL (FIG. 2). MVL is the predominant cyclopropenoid fatty acid in wild-type cottonseed oil.
The relatively high level of saturated fatty acids, mainly palmitic acid, in cottonseed oil compared to oils from most other temperate zone oilseed crops contributes to the oxidative stability of cottonseed oil by offsetting the greater instability of the other, unsaturated fatty acid components. It also imparts the high melting point required for making such products as margarine and shortening. On the other hand, higher levels of palmitic acid are nutritionally less desirable because of its property of raising LDL-cholesterol in humans, associated with increased risk of cardiovascular heart disease (CHD) (Lindsey et al., Exp. Biol. Med. 195: 261-269, 1990). Except for palm oil, cottonseed contains the highest palmitic acid level (26%) among the major commodity vegetable oils. Palmitic acid and other shorter chain saturated fatty acids are widely reported to raise total plasma cholesterol and low density lipoprotein cholesterol levels (Kris-Etherton et al., Nutrition-today (USA). 28: 30-38, 1993). Cottonseed oil also contains a high level of linoleic acid which is oxidatively unstable and therefore limits the shelf life of the oil and makes it unsuitable for some food applications.
Conventional cottonseed oil is therefore often processed by partial hydrogenation during which the polyunsaturated linoleic acid is transformed into more stable monounsaturated (oleic) and saturated (stearic) fatty acids. Partial hydrogenation results in a number of structural changes to a fraction of the fatty acids, including the shifting of a double bond. This may lead to the production of trans fatty acids (TFA) which are isomers of the naturally occurring unsaturated fatty acids. In most naturally occurring unsaturated fatty acids such as oleic acid, the two hydrogen atoms joined to the double-bonded carbon atoms are on the same side of the double bonds of the carbon chain, and this is termed a cis double bond. However, partial hydrogenation re-configures some of the double bonds so that the two hydrogen atoms are on opposite sides of the carbon-carbon double bond, as it is the lower energy form, and this is termed the trans configuration. Thus partial hydrogenation produces trans fatty acids, such as elaidic acid from oleic acid. Oleic and elaidic acids contain the same number of atoms (C18:1), with a double bond in the same location, but it is the conformation of the double bond that sets them apart. TAG containing elaidic acid, with the trans double bond configuration, has a much higher melting point than oleic acid. In recent years TFA have been increasingly recognized to have significant LDL-cholesterol raising and HDL-cholesterol lowering properties and therefore increases the risk of cardiovascular disease based on evidence derived from epidemiologic and clinical studies (Oomen et al., Lancet 357: 746-751, 2001; Mozaffarian et al., N. Engl. J. Med. 354: 1601-1613, 2006). Partial hydrogenation also converts cyclopropanoic or cyclopropenoic fatty acids to branched chain fatty acids by opening up the cyclopropane ring, producing a branched fatty acid with a additional methyl group attached to C9 or C10 of the fatty acid carbon chain.
Compared with polyunsaturated fatty acids, oleic acid is more stable towards oxidation both at ambient storage temperatures and at the high temperatures used in cooking and frying of food. Studies with a number of vegetable oils such as safflower and soybean oils indicate that high-oleic vegetable oils are slower to develop rancidity during storage, or to oxidatively decompose during frying or other use, compared to oils that contain high amounts of polyunsaturated fatty acids (Fuller et al., J. Food Sci. 31: 477-480, 1966; Mounts et al., J. Am. Oil Chem. Soc. 65: 624-628, 1998).
As mentioned above, cottonseed oil is also characterized by the presence of small amounts of malvalic, sterculic and dihydrosterculic acids, a group of cyclic propane fatty acids derived from oleic acid. It is known that malvalic and sterculic acids are potent inhibitors of animal Δ9-stearoyl-CoA desaturase. Although the CPA and CPE fatty acids are not stable and are mostly eliminated during oil processing, particularly by hydrogenation, the residual oil in the meal and the whole cottonseed used in the feed industry could exert negative effects on animal health. Feeding farmed animals with excess amounts of cottonseed is thought to possibly cause a number of health problems for animals and may affect the quality of animal products, such as the hardening of fats in egg yolk and milk (Johnson et al., Nature 214: 1244-1245, 1967; Roehm et al., Lipids 5: 80-84, 1970). Methods have been developed to inactivate cyclopropenoid fatty acids through specialised partial hydrogenation processes. Merker and Mattil, 1965 reported a hydrogenation process in which malvalic and sterculic acids were selectively reduced to their dihydro or tetrahydro derivatives, by means of a nickel catalyst, without significant reduction of the linoleic acid or trans acid formation. Hutchins et al., Journal of American Oil Chemists Society 45: 397-399, 1968 showed selective hydrogenation of the cyclopropenoid groups in cottonseed oil by means of a packed-bed reactor and nickel catalysts under milder conditions. However, these hydrogenation processes add additional costs for processing of the oil and are not desirable.
In the 1970's, the cotton breeding program of the Acala SJ series in California (Cherry, 1983 (supra)) reduced palmitic acid from 23.3 to 22.7%, increased oleic acid from 16.6% to 17.3% and reduced total cyclic fatty acids from 0.9% to 0.8% in cottonseed oil. However, compared to achievements in other oilseed crops, these changes were only minor, reflecting the narrow genetic base of elite cotton varieties as a result of persistent selection on traits other than oil quality.
Four different cDNAs encoding FAD2 were isolated from cotton (Liu et al., Australian Journal of Plant Physiology 26: 101-106, 1999a; Liu et al., Plant Physiol. 120: 339, 1999b; Pirtle et al., Biochim. Biophys. Acta 1522: 122-129, 2001, all herein incorporated by reference), among which ghFAD2-1 was determined to play a major role in the production of linoleic acid in cottonseed oil. Analysis of gene expression suggested that the ghFAD2-1 gene was specifically expressed in developing seeds, with maximal expression during the middle maturity stage of seed development (Liu et al., 1999a (supra)).
U.S. Pat. No. 6,974,898 (herein incorporated by reference) describes the generation of cottonseed oil containing up to 77% oleic acid by downregulation of microsomal Δ12 desaturase (FAD2) by RNAi methods.
Chapman et al., J. Am. Oil Chem. Soc. 78: 941-947, 2001, herein incorporated by reference, transformed cotton plants with a construct encoding a non-functional, mutant allele of fad2, obtained from rapeseed, and expressed from a seed-specific phaseolin promoter in sense orientation. About half of the 43 transgenic lines that were generated showed slightly increased oleic acid content in seedoil, ranging from 24-30%. In subsequent generations, seeds with higher levels of oleic acid up to 40% were identified.
Arabidopsis fatB mutants which had seedoil with reduced palmitic acid levels showed reduced vegetative growth under normal temperatures (Bonaventure et al., Plant Cell 15: 1020-1033, 2003). However, in previous studies with antisense constructs to down-regulate AtFatB in Arabidopsis, using the CaMV35S promoter to express the antisense RNA, a substantial reduction of palmitic acid levels was observed only in flowers and seeds, not in leaves and roots, and there was no obvious visual phenotype (Dormann et al., Plant Physiol. 123: 637-644, 2000, herein incorporated by reference).
In cotton, as in many temperate plants, FatB enzymes appear to be encoded by a multigene family. Yoder et al., Biochimica et Biophysica Acta 1446: 403-413, 1999, herein incorporated by reference, isolated a genomic DNA sequence which was thought to encode a acyl-ACP thioesterase in cotton.
Wilson et al., J. Am. Oil Chem. Soc. 78: 335-340, 2001 and Buhr et al., Plant J. 30: 155-163, 2002, both herein incorporated by reference, used antisense and ribozyme approaches, respectively, to reduce palmitic acid in soybean seeds.
A CPA-FAS gene was recently isolated from Sterculia foetida using an EST based approach (Bao et al., Proc. Natl. Acad. Sci. U.S.A. 99: 7172-7177, 2002). Two EST sequences from G. hirsutum were identified as being differentially expressed by infection of cotton roots/hypocotyls following inoculation of Fusarium (Dowd et al., Molecular Plant-Microbe Interactions. 17: 654-667, 2004, herein incorporated by reference).
There is therefore a need for improved cottonseed oil.